The objective of this project is to continue studies to determine what chemical and ultrastructural modifications of the outer membrane (OM) of Pseudomonas aeruginosa accompanies the acquisition of resistance to increasing concentrations of selected antibiotics, both in a model system and in clinical isolates. A model system for the development of resistance to the antibiotic polymyxin B has been developed in P. aeruginosa (Gilleland and Murray, J. Bacteriol. 125: 267-281, 1976). The OM of cells adapted to grow in the presence of 6000 units of polymysin B per ml was shown to be ultrastructually altered. Cell envelopes from polymyxin-resistant isolates have been shown to have lost lipopolysaccharide and major OM proteins (Gilleland and Lyle, J. Bacteriol. 138: 839-845, 1979). Isolates resistant to increasing concentrations of polymyxin B will be examined by freeze-etching for the presence of an accompanying progressive ultrastructural alteration of the OM. The ability of polymyxin-resistant cells to bind the antibiotic will be determined using radioactively labeled polymyxin. Studies which are in progress of lipid alterations in the resistant strains will be completed. OM proteins will be purified for use in reconstitution studies in which phospholipid vesicles merge with the OM resistant cells in the attempt to restore polymyxin-sensitivity to the cells. These studies will yield a much clearer understanding of the role of the OM in mediating antibiotic resistance through an impermeability mechanism.